Overall, the expertise of the Mass Spectrometry Unit is being widely used to further the research of multiple groups within the CCR. In FY2016, the unit collaborated in 44 different projects, with more than 1750 samples processed and analyzed. These projects are being performed in collaboration with 32 different investigators. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, ubiquitination, acetylation, methylation, and hydroxylation, to better understand signal transduction, protein regulation, and the effects of small molecule inhibitors. The resource is also being used to identify protein interactors of both proteins and nucleic acids, including identification of those that change following post-translational modification. These studies help provide critical insight into protein function and regulation. Mass spectrometry is additionally being used extensively for large-scale quantitative proteomics projects. In these, labeled or label-free methods are used to comprehensively identify whole proteomes, such as the protein composition of conditioned media, biological fluids (i.e., aqueous humor), a subcellular organelle or vesicle (e.g., mitochondria and exosomes), or a whole cell. Additionally, we are collaborating on a global quantitative phosphoproteome study, in which the global level of phosphorylation is compared. These discovery-oriented studies, which are sample- and time-intensive, provide broad information for defining new hypotheses and provide new insight into global protein activities and cellular responses. Within the last year, the Mass Spectrometry Unit has begun adding crosslinking mass spectrometry (XL-MS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to the techniques available to CCR collaborators. In a XL-MS experiment, mass spectrometry is used to identify residues within separate peptides that are crosslinked together, providing information about which parts of a protein or protein complex are within a specified distance. Multiple crosslinkers of different legnths can be used to map the protein complexes. In an HDX-MS experiment, mass spectrometry is used to determine the deuterium uptake of proteins incubated with deuterium in the native state. By analyzing the change in deuterium uptake over time, protein dynamics and conformational changes can be probed. Furthermore, by comparing the exchange profile of a protein in different conditions or in the presence of a binding partner or ligand, more detailed information about changes in the protein conformation can be obtained. In the past year, three collaborative studies have been published, and several other projects have been completed and the corresponding manuscripts are under review. The first study, a collaboration with Dr. Michael Gottesman, Laboratory of Cell Biology, investigated the molecular basis for observed changes in bioluminescence imaging (BLI) signal intensity when cyclodextrin was co-injected as an excipient for molecular imaging studies. Cyclodextrins are a family of biocompatible cyclic oligosaccharides that form a barrel shape and display relatively high solubility due to the presence of hydrophilic hydroxyl groups on their exterior surface. As their interior is relatively nonpolar, they can non-covalently associate with lipophilic small molecules, effectively increasing their apparent solubility. While investigating the formulation of an ABCG2 inhibitor in a formulation containing cyclodextrin, the Gottesman lab observed unexpected changes in BLI signal intensity compared to control mice injected with D-luciferin dissolved in other formulations. Using mass spectrometry, we demonstrated that the luciferin actually covalently modified the cyclodextrin, leading to a reduction in bioluminescent signal in animals using this formulation. Co-administration of several experimental therapeutic ABC transporter inhibitors led to an increase in BLI signal due to the displacement of the luciferin from the cyclodextrin. Thus, the mass spectrometry analysis was crucial for demonstrating the mechanism by which the cyclodextrin led to changes in the luciferin signaling. These studies, published in Molecular Imaging, have critical implications for the design of bioluminescence-based imaging studies. In the second study, mass spectrometry was used to identify protein mediators of the cellular functions of ASAP1, an Arf GAP that associates with and regulates actin-based structures. Mass spectrometry analysis of the protein interactors of an ASAP1 construct containing the BAR and PH domains adsorbed to large unilamellar vesicles (LUVs) identified myosin-9 as a dominant interactor. Significantly less myosin-9 was found to bind to LUVs alone or the PH domain plus LUVs, indicating that the interaction was specific to the BAR domain. Myosin-9 is part of cytoplasmic nonmuscle myosin 2A (NM2A), an F-actin binding ATPase that functions as a molecular motor in cellular events that involve force or translocation. NM2A cross-links F-actin and the complex of NM2A and F-actin generates contractility necessary for cellular functions including regulation of focal adhesions, cell spreading, and cell migration. Intracellularly, NM2A and ASAP1 were observed to colocalize at the junction of focal adhesions and stress fibers and more extensively at circular dorsal ruffles (CDRs). Further experiments to determined that ASAP1 and NM2A affected a common set of actin structures and cell behaviors that depend on actin remodeling, indicating that ASAP1 is a regulator of NM2A. Our mass spectrometry analysis was crucial for initiating these studies, as it was the first observation that myosin-9 was able to interact with the ASAP1 BAR domain. This research was performed in collaboration with Dr. Paul Randazzo, Laboratory of Cellular and Molecular Biology, and published in the Journal of Biological Chemistry. Finally, working with Dr. Deborah Hinton, National Institute of Diabetes and Digestive and Kidney Diseases, mass spectrometry was used to help establish a model for sigma appropriation, the process whereby bacteriophage T4 hijacks the sigma subunit of host E. coli RNA polymerase (RNAP) to redirect RNAP from the expression of host genes to T4 promoter DNA. During sigma appropriation, there is conformational rearrangement of the E. coli sigma protein by binding of the phage-encoded proteins MotA and AsiA. Despite understanding how pieces of MotA, AsiA and RNAP connect during sigma appropriation and the existence of structures of parts of the complex, there is not a structure of the entire complex or how it interacts with DNA. Using multiple experimentally determined 3D structures, molecular modeling, and biochemical observations indicating the position of the proteins relative to each other and to the DNA, a structural model of the multimeric complex was developed. Crosslinking analyses were used to help constrain the model, including crosslinking-mass spectrometry experiments that identified a critical crosslink between the sigma and beta' proteins. The mass spectrometry analysis provided strong experimental support for the model, which provides critical insights into sigma appropriation and provides insight into how a mutation within the beta subunit of RNAP far removed from AsiA or MotA impairs sigma appropriation. The results of this study are in press in Nucleic Acids Research.